CN107760607A - A kind of aspergillus niger mutant strain and its application - Google Patents

A kind of aspergillus niger mutant strain and its application Download PDF

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CN107760607A
CN107760607A CN201610701711.0A CN201610701711A CN107760607A CN 107760607 A CN107760607 A CN 107760607A CN 201610701711 A CN201610701711 A CN 201610701711A CN 107760607 A CN107760607 A CN 107760607A
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fermentation
enzyme
aspergillus niger
fermentation process
culture
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CN107760607B (en
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徐晓东
周利伟
王华明
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Weifang KDN Biotechnology Co., Ltd.
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Qingdao Vland Biotech Group Co Ltd
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    • C12N9/2477—Hemicellulases not provided in a preceding group
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    • C12R2001/685—Aspergillus niger

Abstract

The present invention relates to microorganism field, more particularly to a kind of aspergillus niger mutant strain and its application.Shake flask fermentation and ferment tank result are shown, compared to starting strain, the aspergillus niger mutant strain significantly improves the yield of non-starch polysaccharide enzyme, it can be widely applied to the production of non-starch polysaccharide enzyme, so as to advantageously reduce the production cost of non-starch polysaccharide enzyme, promote its popularization and application in field of fodder.

Description

A kind of aspergillus niger mutant strain and its application
Technical field
The present invention relates to microorganism field, more particularly to a kind of aspergillus niger mutant strain and its application.
Background technology
SNSP (non-starchpolysaccharides, NSP) is by a variety of monose and alditol in plant tissue Acid is formed by connecting through glycosidic bond, is mostly the chain structure for having branch, is often combined together with inorganic ions and protein, is thin The main component of cell wall, it is generally more difficult to be hydrolyzed by the digestive ferment of nonruminant secretion.SNSP master in common feed If araboxylan, beta glucan and cellulose.Contain a small amount of SNSP, oat and barley in corn and sorghum In soluble non-starch polysaccharide be mainly beta glucan.Contain a small amount of pectin polysaccharide in cereal, in addition to rice, in other plant Do not find.Cereal byproduct contains substantial amounts of cell wall constituent, as rice bran contains about 20%-25% SNSP, The mainly araboxylan and cellulose of equivalent.
Non-starch polysaccharide enzyme then based on a variety of glycosidases, is made by the anti-nutrition for eliminating the SNSP in feed With animal being improved to the utilization rate of feed nutrient, when SNSP composition one in the suitable proportion of these enzymatic activitys and feed During cause, optimal using effect can be obtained.Non-starch polysaccharide enzyme includes cellulase, zytase, 1,4 beta-glucanase, β-sweet dew Dextranase, pectase etc..
Cellulase can crack rich fibrous cell membrane, discharge the nutriments such as protein, starch that it includes Come and be used, while can be again the reduced sugar that can be digested and assimilated by livestock and poultry body by fiber degradation, so as to improve feed profit With rate.Produce cellulase microbe research it is more be fungi, bacterium and actinomyces are studied seldom.It is current to be used for producing The microorganism of cellulase is mainly trichoderma, aspergillus niger, mould and head mold, in addition, paint spot is mould, instead works as animal rumens bacterium, thermophilic fibre Dimension bacterium, single armful of bacterium of production yellowish fiber, side armful bacterium, slime bacteria, fusiform bud pole bacterium etc. can also produce cellulase.
Zytase is the single-minded digestive enzyme of xylan, belongs to hydrolase, including endo-xylanase, circumscribed xylan Three kinds of enzyme and xylosidase.Research both at home and abroad on trichoderma, aspergillus, bacterium production zytase ability is more, commercialization now The bacterial strain of production zytase be mainly trichoderma and aspergillus.
1,4 beta-glucanase can degrade β -1 in beta glucan molecule, 3 and β-Isosorbide-5-Nitrae glycosidic linkage, be allowed to be degraded to small molecule, Hydrophily and viscosity are lost, changes the characteristic, the activity of digestive ferment, the effect ring of enteric microorganism of nonruminant intestinal contents Border etc..The microorganism of 1,4 beta-glucanase is secreted, one kind is bacterium, and another kind of is fungi, and fungi mainly has Kang Shi based on mould Trichoderma, trichoderma reesei, Ni Shi trichodermas, Trichoderma viride, aspergillus oryzae, mucor hiemalis, aspergillus niger etc..
Pectase is the common name for the enzyme for decomposing pectin, an and multienzyme complex, it generally includes protopectinase, pectin Three kinds of cruel hydrolase of first, pectinesterase enzymes.The synergy of these three enzymes enables pectic substance to decompose completely.Industrial production pectin The strain of enzyme is mainly mould, and conventional strain has aspergillus wentii, apple mould, aspergillus niger, white rot pyrenomycetes, aspergillus oryzae, yeast etc..
Mannase is a kind of hydrolysis of hemicellulose enzyme, with internal-cutting way degrade β -1,4 glycosidic bonds, catabolite it is non- Reduction end is mannose, and its substrate specificity includes glucomannan, galactomannans and beta-mannase etc..It is not only Enteron aisle viscosity can be reduced, promotes the digestion and absorption of nutriment, but also the β-dew glycan pair being rich in beans can be eliminated The interference of glucose absorption, the energy digestibility of grouts especially dregs of beans is greatly improved；Meanwhile moved after with the addition of mannase The resistance and regularity of thing all increase.
Mainly by biological fermentation process, it is more that what people found at present can produce various non-starch for the production of non-starch polysaccharide enzyme The bacterial strain of carbohydrase has Trichoderma viride, red trichoderma, aspergillus niger and strand mould etc..Microorganism wherein for producing cellulase It is fungi to study more, bacterium and actinomyces is studied seldom, and the current microorganism for being used for producing cellulase is mainly wood Mould, aspergillus niger, mould and head mold.Research both at home and abroad on trichoderma, aspergillus, bacterium production zytase ability is more, present business The bacterial strain of the production zytase of industry is mainly trichoderma and aspergillus.The microorganism of 1,4 beta-glucanase is secreted, one kind is bacterium, with Based on bacillus, mainly there are bacillus subtilis, bacillus amyloliquefaciens, bacillus licheniformis etc.；Another kind of is fungi, with Based on mould, mainly there are koning trichoderma, trichoderma reesei, Ni Shi trichodermas, Trichoderma viride, aspergillus oryzae, mucor hiemalis, aspergillus niger etc.. The strain of industrial production pectase is mainly mould, and conventional strain has aspergillus wentii, apple mould, aspergillus niger, white rot pyrenomycetes, rice Aspergillus, yeast etc..
China's main energetic feed resource shortage, and SNSP significantly limit cereal and its byproduct in feed In application, therefore be badly in need of for different diet backgrounds exploitation high yield non-starch polysaccharide enzyme bacterial strain, reduce non-starch polysaccharide enzyme Applied to the cost of feed industry, effectively alleviate shortage of resources problem.
The content of the invention
In view of this, the invention provides a kind of aspergillus niger mutant strain of high yield non-starch polysaccharide enzyme and its application.This Invention obtains the aspergillus niger mutant strain of plant height production non-starch polysaccharide enzyme by the method for ultraviolet mutagenesis, can increase substantially non- The expression quantity of starch-polysaccharides enzyme, it can be widely applied to the production of non-starch polysaccharide enzyme.
In order to realize foregoing invention purpose, the present invention provides following technical scheme：
The invention provides one plant of aspergillus niger, its deposit number is CCTCC NO:M2016362.
The present invention obtains one plant of NSP production of enzyme highest mutant strain by the method for ultraviolet mutagenesis, is named as aspergillus niger NSP-5 (Aspergillus niger NSP-5), xylanase activity 106u/ml in the bacterial strain shake flask fermentation supernatant, than going out Bacterium germination improves 96%；Cellulose enzyme activity 4.4u/ml, 91% is improved than going out bacterium germination；1,4 beta-glucanase enzyme activity 41.4u/ml, 55% is improved than going out bacterium germination；Mannase enzyme activity 1.9u/ml, 175% is improved than going out bacterium germination.Ferment tank experiment knot Fruit shows：Mutant bacteria aspergillus niger NSP-5 fermented supernatant fluid xylanase activity 205u/ml, 107% is improved than going out bacterium germination； Cellulose enzyme activity 17.6u/ml, 69% is improved than going out bacterium germination；1,4 beta-glucanase enzyme activity 67.7u/ml, is improved than going out bacterium germination 78%；Mannase enzyme activity 3.9u/ml, 160% is improved than going out bacterium germination, each component expression quantity amplification compared with shaking flask Substantially.Present invention applicant is on July 4th, 2016 by above-mentioned mutant strain aspergillus niger NSP-5 (Aspergillus niger NSP-5 the China typical culture collection center of Wuhan, China Wuhan University) is preserved in, deposit number is CCTCC NO: M2016362。
Present invention also offers application of the aspergillus niger in fermenting and producing non-starch polysaccharide enzyme.
In some specific embodiments of the present invention, the non-starch polysaccharide enzyme is zytase, cellulase, β-Portugal Mixture more than one or both of dextranase and mannase.
Present invention also offers a kind of fermentation process for producing non-starch polysaccharide enzyme, using the aspergillus niger as fermentation strain.
In some specific embodiments of the present invention, the fermentation process includes shake flask fermentation and ferment tank；
The fermentation medium of the fermentation process includes the mass parts of wheat bran 2；The mass parts of lactose 0.3；The mass of corn steep liquor 2 Part；The mass parts of ammonium sulfate 0.5；The mass parts of potassium dihydrogen phosphate 0.35；The mass parts of dipotassium hydrogen phosphate 0.75；The matter of epsom salt 0.03 Measure part.
In some specific embodiments of the present invention, the fermentation temperature of shake flask fermentation described in the fermentation process is 30 DEG C, fermentation time 5d.
In some specific embodiments of the present invention, ferment tank described in the fermentation process includes Shaking culture And fermentation tank culture；
The culture medium of the Shaking culture includes glucose 10-30g/L, potato 100-200g/L；
The condition of the Shaking culture is in 30 DEG C, 200rpm shaking table cultures 20h.
In some specific embodiments of the present invention, the condition of fermentation tank culture is described in the fermentation process： 30 ± 1 DEG C, pH value is 5.0 ± 0.2, and under conditions of mixing speed is 600rpm, fermentation strain is inoculated in such as claim 5 institute After cultivating 24h in the fermentation medium stated, xylose+glucose reversion syrup is added, it is 30-40% to control dissolved oxygen, and ferment 160h.
In some specific embodiments of the present invention, xylose described in the fermentation process+glucose reversion syrup Preparation method is：Take xylose 25%, glucose 25%, 85% phosphoric acid 5%v/v, 121 DEG C of processing 30min.
Present invention also offers the non-starch polysaccharide enzyme that the fermentation of described fermentation process obtains；The non-starch polysaccharide enzyme is Zytase, cellulase, the mixture of one or both of 1,4 beta-glucanase and mannase above.
Shake flat experiment result is shown：It is 54u/ml to go out xylanase activity in bacterium germination aspergillus niger NSP-1 fermented supernatant fluids, fiber Plain enzyme activity is 2.3u/ml, and 1,4 beta-glucanase enzyme activity is 26.6u/ml, and mannase enzyme activity is 0.69u/ml.The present invention obtains One plant of NSP production of enzyme highest mutant strain, it is named as aspergillus niger NSP-5 (Aspergillus niger NSP-5), the bacterial strain Xylanase activity 106u/ml in fermented supernatant fluid, 96% is improved than going out bacterium germination；Cellulose enzyme activity 4.4u/ml, than setting out Bacterium improves 91%；1,4 beta-glucanase enzyme activity 41.4u/ml, 55% is improved than going out bacterium germination；Mannase enzyme activity 1.9u/ml, 175% is improved than going out bacterium germination, achieves unexpected technique effect.Present invention applicant will be upper on July 4th, 2016 State mutant strain aspergillus niger NSP-5 (Aspergillus niger NSP-5) and be preserved in the Chinese allusion quotation of Wuhan, China Wuhan University Type culture collection, deposit number are CCTCC NO:M2016362.
Ferment tank experimental result is shown：The fermented supernatant fluid xylanase activity for going out bacterium germination is 99u/ml, cellulose Enzyme enzyme activity is 10.4u/ml, and 1,4 beta-glucanase enzyme activity is 37.9u/ml, and mannase enzyme activity is 1.5u/ml.The black song of mutant bacteria Mould NSP-5 fermented supernatant fluid xylanase activity 205u/ml, 107% is improved than going out bacterium germination；Cellulose enzyme activity 17.6u/ Ml, 69% is improved than going out bacterium germination；1,4 beta-glucanase enzyme activity 67.7u/ml, 78% is improved than going out bacterium germination；Mannase enzyme 3.9u/ml living, 160% is improved than going out bacterium germination, and each component expression quantity amplification compared with shaking flask is obvious, is achieved and is expected not The technique effect arrived.
Biological deposits explanation
Biomaterial aspergillus niger NSP-5, Classification And Nomenclature：Aspergillus niger NSP-5 (Aspergillus niger NSP-5), in On July 4th, 2016 China typical culture collection center for being preserved in Wuhan, China Wuhan University, collection address is：Lake Bei Sheng Wuchang Luo Jia Shan Wuhan University is in the school；Deposit number is CCTCC NO:M2016362.
Embodiment
The invention discloses a kind of aspergillus niger mutant strain and its application, those skilled in the art can be used for reference in this paper Hold, be suitably modified technological parameter realization.In particular, all similar replacements and change are to those skilled in the art For be it will be apparent that they are considered as being included in the present invention.The method of the present invention and application are by preferably implementing Example is described, related personnel substantially can not depart from present invention, in spirit and scope to method described herein and Using being modified or suitably change with combining, to realize and using the technology of the present invention.
One aspect of the present invention is related to a kind of new mutant strain aspergillus niger NSP-5 (Aspergillus niger NSP-5), It is in the China typical culture collection center for being preserved in Wuhan, China Wuhan University on July 4th, 2016, deposit number CCTCC NO:M2016362。
The culture medium each component and its mass ratio used in the fermentation process is wheat bran 2%；Lactose 0.3%；It is beautiful Rice ＆ peanut milk 2%；Ammonium sulfate 0.5%；Potassium dihydrogen phosphate 0.35%；Dipotassium hydrogen phosphate 0.75%；Epsom salt 0.03%.
The mutant strain aspergillus niger NSP-5 that present invention screening obtains, Shaking culture is after 5 days, xylan in fermented supernatant fluid Enzyme enzyme activity is 106u/ml, and 96% is improved than going out bacterium germination；Cellulose enzyme activity is 4.4u/ml, and 91% is improved than going out bacterium germination； 1,4 beta-glucanase enzyme activity is 41.4u/ml, and 55% is improved than going out bacterium germination；Mannase enzyme activity is 1.9u/ml, than going out bacterium germination 175% is improved, achieves unexpected technique effect.
Ferment tank experimental result is shown：The fermented supernatant fluid xylanase activity for going out bacterium germination is 99u/ml, cellulose Enzyme enzyme activity is 10.4u/ml, and 1,4 beta-glucanase enzyme activity is 37.9u/ml, and mannase enzyme activity is 1.5u/ml.The black song of mutant bacteria Mould NSP-5 fermented supernatant fluid xylanase activity 205u/ml, 107% is improved than going out bacterium germination；Cellulose enzyme activity 17.6u/ Ml, 69% is improved than going out bacterium germination；1,4 beta-glucanase enzyme activity 67.7u/ml, 78% is improved than going out bacterium germination；Mannase enzyme 3.9u/ml living, 160% is improved than going out bacterium germination, and each component expression quantity amplification compared with shaking flask is obvious, is achieved and is expected not The technique effect arrived.
The aspergillus niger mutant strain can be widely applied to the production of non-starch polysaccharide enzyme, so as to advantageously reduce non-starch The production cost of polysaccharase, promote popularization and application of the non-starch polysaccharide enzyme in field of fodder.
The routine techniques and method that the present invention has used genetic engineering and biology field uses, such as MOLECULAR CLONING:A LABORATORY MANUAL, 3nd Ed. (Sambrook, 2001) and CURRENT Described method in PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, 2003).These general bibliography Provide definition well known by persons skilled in the art and method.But those skilled in the art can be described in the present invention Technical scheme on the basis of, using this area other conventional method, experimental program and reagents, and be not limited to of the invention specific The restriction of embodiment.
Raw materials used and reagent can be bought by market in aspergillus niger mutant strain provided by the invention and its application.
With reference to embodiment, the present invention is expanded on further：
Embodiment 1 goes out bacterium germination aspergillus niger shake flask fermentation and Enzyme activity assay
Bacterium germination aspergillus niger NSP-1 (Aspergillus niger NSP-1) will be gone out, and (bacterial strain is by one of inventor Xu Xiao East is in January, 2015 screened from Qingdao City's Laoshan District soil) it is seeded to fresh PDA plate, 30 DEG C of culture 5-7d.
The fungus block of 2cm × 2cm sizes is extracted, is seeded to 50ml liquid submerged cultures base (wheat bran 2%；Lactose 0.3%；Corn steep liquor 2%；Ammonium sulfate 0.5%；Potassium dihydrogen phosphate 0.35%；Dipotassium hydrogen phosphate 0.75%；Epsom salt 0.03%) fermented in, 30 DEG C are cultivated 5 days；Zymotic fluid is centrifuged, the supernatant of acquisition is crude enzyme liquid；To supernatant point Carry out not cellulase, dextranase, zytase enzyme, the enzyme activity determination of mannase.As a result show, go out the black song of bacterium germination Xylanase activity is 54u/ml in mould NSP-1 fermented supernatant fluids, cellulase activity 2.3u/ml, and 1,4 beta-glucanase enzyme activity is 26.6u/ml, mannase enzyme activity are 0.69u/ml.
1. cellulase activity detects
(1) definition of cellulose enzyme activity unit
It is per minute from the carboxymethyl cellulose that concentration is 5mg/ml under the conditions of pH is 4.80 (neutrality is pH6.0) at 50 DEG C Enzyme amount in sodium solution required for degraded release 1umol reduced sugars is a unit of activity (IU), and reduced sugar is with glucose equivalent.
(2) enzyme activity determination method
(2.1) drafting of standard curve：
Take 8 test tube accordings to the form below to add 1.5mlDNS reagents after adding related test solution, fully shake up, put anti-in boiling water bath Answer 5min.Room temperature is rapidly cooled to, 5.0ml is settled to water, by the use of No. 0 test tube test solution as control, it is surveyed under 540nm wavelength The absorbance of its each test tube test solution.It is that abscissa draws standard curve with (glucose content/100) using absorbance as ordinate.
Test tube number 0 1 2 3 4 5 6 7
Buffer solution addition (ul) 1000 990 985 980 975 970 965 960
Glucose standard addition (ul) 0 10 15 20 25 30 35 40
Glucose content (ug) 0 100 150 200 250 300 350 400
(2.2) enzyme activity determination：
Four test tubes are taken respectively to add 0.5ml CMC substrates, 50 DEG C of water-bath preheating 5min, first three branch are together with enzyme liquid to be measured Sample tube, the 4th is blank tube.0.5ml prepare liquids, and timing are respectively added in first three branch test tube, is reacted in 50 DEG C of water-baths 15min。
1.5ml DNS reagents are respectively added after having reacted in first three branch test tube.Then added successively to each blank tube 1.5mLDNS, 0.5ml enzyme liquid to be measured is finally added into blank tube successively.
After taking out and shaking up three test tubes, 5min is reacted in boiling water bath.Room temperature is rapidly cooled to, 5.0ml is arrived surely with water. Using blank cuvette test solution as the absorbance to impinging upon determination sample pipe test solution under 540nm wavelength conditions, absorbance 0.25~ It is advisable between 0.30.If it need to not change extension rate in this scope and resurvey.
Enzyme activity calculation formula：
Enzyme activity (IU/ml or IU/g)=(value/180/15/0.5 such as glucose) × n
In formula：180-- glucose is converted into micromole from microgram
The reaction time of 15-- prepare liquid and substrate
0.5-- adds the enzyme liquid amount to be measured of reaction
The extension rate of n-- enzyme sample
2. activity of beta-glucanase detects
(1) definition of 1,4 beta-glucanase enzyme-activity unit
Under conditions of 37 DEG C, pH value are 5.5, per minute degraded from the beta glucan solution that concentration is 4mg/ml is released Enzyme amount required for putting 1 μm of ol reduced sugar is an enzyme activity unit U.
(2) enzyme activity determination method
(2.1) drafting of standard curve：
Acetate-sodium acetate buffer 4.0ml is drawn, adds DNS reagent 5.0ml, boiling water bath heating 5min.With originally Room temperature is water-cooled to, 25.0ml is settled to water, standard blank sample is made.Draw respectively glucose solution 1.00ml, 2.00ml, 3.00ml, 4.00ml, 5.00ml, 6.00ml and 7.00ml, 100ml is settled to acetate-sodium acetate buffer respectively, matched somebody with somebody It is 0.10mg/ml, 0.20mg/ml, 0.30mg/ml, 0.40mg/ml, 0.50mg/ml, 0.60mg/ml, 0.70mg/ that concentration, which is made, Ml glucose standards solutions.
Each 2.00ml of glucose standards solution (do two parallel) of above-mentioned concentration series is drawn respectively, is added separately to carve Spend in test tube, then be separately added into 2.0ml acetate-sodium acetate buffers and 5.0ml DNS reagents.Electromagnetic viscosimeter 3s~5s, boiling Heating water bath 5min.Then room temperature is cooled to running water, then 25ml is settled to water.Returned to zero by control of standard blank sample, Absorbance OD values are determined at 540nm.
It is X-axis by Y-axis, absorbance OD values of concentration of glucose, draws standard curve.New DNS reagents of preparing are both needed to every time Repaint standard curve.
(2.2) enzyme activity determination：
Draw 10.0ml beta glucan solution, 37 DEG C of balance 20min.
10.0ml is drawn by the enzyme liquid suitably diluted, 37 DEG C of balance 10min.
2.00ml is drawn by the enzyme liquid (having been subjected to 37 DEG C of balances) suitably diluted, is added in scale test tube, adds 5ml DNS reagents, electromagnetic viscosimeter 3s.Then 2.0ml beta glucan solution, 37 DEG C of balance 30min, boiling water bath heating are added 5min.With room temperature is originally water-cooled to, water is added to be settled to 25ml, electromagnetic viscosimeter 3s~5s.Using standard blank sample as blank control, Absorbance A is determined at 540nmB。
2.00ml is drawn by the enzyme liquid (having been subjected to 37 DEG C of balances) suitably diluted, is added in scale test tube, adds 2.0ml beta glucans solution (has been subjected to 37 DEG C of balances), electromagnetic viscosimeter 3s, 37 DEG C of accurate insulation 30min.Add 5.0ml DNS Reagent, electromagnetic viscosimeter 3s, with enzymolysis reaction.Boiling water bath heats 5min, with room temperature is originally water-cooled to, adds water to be settled to 25ml, electromagnetic viscosimeter 3s.Using standard blank sample as blank control, absorbance A is determined at 540nmE。
Enzyme activity calculation formula：
XD=[(AE-AB)×K+C0]×N×1000÷(M×t)
In formula：XDFor the vigor of zytase in dilution enzyme liquid, U/ml；AEFor the absorbance of enzyme reaction solution；ABFor enzyme blank The absorbance of liquid；K is the slope of standard curve；C0For the intercept of standard curve；M be xylose molal weight, 150.2g/mol； T is the enzyme digestion reaction time, min；N is enzyme liquid extension rate；1000 be transforming factor, 1mmol=1000 μm of ol.
3. Xylanase activity detects
(1) definition of xylanase activity unit
It is per minute to discharge 1 μm of ol from the xylan solution that concentration is 5mg/ml under conditions of 37 DEG C, pH value are 5.5 Enzyme amount required for reduced sugar is an enzyme activity unit U.
(2) enzyme activity determination method
The xylan substrate (preparation of pH5.5 acetic acid-sodium acetate buffer solutions) for taking 2ml concentration to be 1%, is added to colorimetric cylinder In, 37 DEG C of balance 10min, add the acid that 2ml suitably dilutes through pH5.5 acetic acid-sodium acetate buffer solutions and balanced through 37 DEG C Property zytase enzyme liquid, is mixed in 37 DEG C of accurate insulation reaction 30min.Reaction terminate after, add 5ml DNS reagents, mix with Terminating reaction.Then boiling water bath boils 5min, with room temperature is originally water-cooled to, adds distilled water to be settled to 25ml, after mixing, with mark Quasi- blank sample is blank control, and light absorption value A is determined at 540nmE。
Enzyme activity calculation formula：
XD=[(AE-AB)×K+C0]×N×1000÷(M×t)
In formula：XDFor the vigor of zytase in dilution enzyme liquid, U/ml；AEFor the absorbance of enzyme reaction solution；ABFor enzyme blank The absorbance of liquid；K is the slope of standard curve；C0For the intercept of standard curve；M be xylose molal weight, 150.2g/mol； T is the enzyme digestion reaction time, min；N is enzyme liquid extension rate；1000 be transforming factor, 1mmol=1000 μm of ol.
4. mannase viability examination
(1) definition of mannase enzyme-activity unit
Under conditions of 37 DEG C, pH value are 5.5, release per minute of being degraded from the mannan solution that concentration is 3mg/ml Enzyme amount required for 1umol reduced sugars is an enzyme activity unit U.
(2) enzyme activity determination method
(2.1) drafting of standard curve：
Acetic acid-sodium acetate buffer solution 4.0ml is drawn, adds DNS reagent 5.0ml, boiling water bath heating 5min.Use running water Room temperature is cooled to, 25.0ml is settled to water, standard blank sample is made.
Mannose solution (5.5) 1.00,2.00,3.00,4.00,5.00,6.00 and 7.00ml are drawn respectively, use second respectively Acid-sodium acetate buffer solution is settled to 100ml, and it is 0.10~0.70mg/ml D-MANNOSE standard liquids to be configured to concentration.
Each 2.00ml of mannose standard liquid (do two parallel) of above-mentioned concentration series is drawn respectively, is added separately to carve Spend in test tube, then be separately added into 2ml acetate-sodium acetate buffers and 5mlDNS reagents.Electromagnetic viscosimeter 3s, boiling water bath heating 5min.Then room temperature is cooled to running water, then 25ml is settled to water.Returned to zero by control of standard blank sample, at 540nm Determine absorbance OD values.
It is X-axis by Y-axis, absorbance OD values of mannose concentration, draws standard curve.New DNS reagents of preparing are both needed to every time Repaint standard curve.
(2.2) enzyme activity determination：
Draw 10.0ml mannan solutions, 37 DEG C of balance 10min.
10.0ml is drawn by the enzyme liquid suitably diluted, 37 DEG C of balance 10min.
2.00ml is drawn by the enzyme liquid (having been subjected to 37 DEG C of balances) suitably diluted, is added in scale test tube, adds 5mlDNS reagents, electromagnetic viscosimeter 3s.Then 2.0ml mannan solutions, 37 DEG C of insulation 30min, boiling water bath heating 5min are added. With room temperature is originally water-cooled to, water is added to be settled to 25ml, electromagnetic viscosimeter 3s.Using standard blank sample as blank control, at 540nm Determine absorbance AB。
2.0ml is drawn by the enzyme liquid (having been subjected to 37 DEG C of balances) suitably diluted, is added in scale test tube, adds 2.0ml mannan solutions (have been subjected to 37 DEG C of balances), electromagnetic viscosimeter 3s, 37 DEG C of accurate insulation 30min.Add 5.0mlDNS examinations Agent, electromagnetic viscosimeter 3s, enzyme digestion reaction.Boiling water bath heats 5min, with room temperature is originally water-cooled to, adds water to be settled to 25ml, electromagnetism Vibrate 3s.Using standard blank sample as blank control, absorbance A is determined at 540nmE。
Enzyme activity calculation formula：
XD=[(AE-AB)×K+C0]×N×1000÷(M×t)
In formula：XDFor the vigor of zytase in dilution enzyme liquid, U/ml；AEFor the absorbance of enzyme reaction solution；ABFor enzyme blank The absorbance of liquid；K is the slope of standard curve；C0For the intercept of standard curve；M be xylose molal weight, 150.2g/mol； T is the enzyme digestion reaction time, min；N is enzyme liquid extension rate；1000 be transforming factor, 1mmol=1000 μm of ol.
The ultraviolet mutagenesis of embodiment 2 screens with mutant bacteria
Determine fatal rate：Above-mentioned aspergillus niger NSP-1 is inoculated in PDA plate, 30 DEG C of culture 5-7d.Treat that bacterium colony surface becomes It is black, when producing a large amount of spores, the sterile water elutions of 5ml are drawn, spore liquid is obtained, is resuspended after centrifugation with sterilized water, uses blood count Plate counts.A 90mm culture dish is taken, (concentration is about 1 × 10 to the spore suspension that addition 5ml has diluted7Individual/mL), add rotor And stirring makes spore liquid be in uniform state on magnetic stirring apparatus.It is the ultraviolet of 9w with power in aseptic superclean bench Lamp irradiates in vertical range 20cm top, irradiates 30s, 45s, 60s, 75s, 90s, 105s, 120s respectively, takes the spore after irradiation Sub- liquid dilutes 10,100,1000 times, takes 100ul to be coated with PDA plate, is counted after 30 DEG C of culture 2-3d, with non-irradiated spore liquid For control, fatal rate is calculated.When wherein irradiating 120s, fatal rate 95%, choose the irradiation time and carry out follow-up Mutagenesis experiments.
Mutagenesis screening：A 90mm culture dish is taken, (concentration is 1 × 10 to the spore suspension that addition 5ml has diluted7), add Rotor and on magnetic stirring apparatus stirring spore liquid is in uniform state.It is 9w's with power in aseptic superclean bench Uviol lamp irradiates in vertical range 20cm top, dilutes 1000 times after irradiating 90s, takes 100ul to be coated with PDA plate, 30 DEG C of trainings Support 2-3d.
200 pieces of PDA plates are coated with altogether, and after 30 DEG C are cultivated 2-3d, each flat board grows 30-50 bacterium colony, first passes through bacterium colony Form, the mutant of short branch is screened, the mutant 156 that picking colony form is smaller, mycelia is fine and close, periphery of bacterial colonies fine hair is shorter It is individual to be inoculated into PDA plate, 30 DEG C of culture 5-7d.Each transformant extracts the fungus block of 2cm × 2cm sizes, is inoculated in 50ml respectively Liquid submerged culture base (wheat bran 2%；Lactose 0.3%；Corn steep liquor 2%；Ammonium sulfate 0.5%；Potassium dihydrogen phosphate 0.35%； Dipotassium hydrogen phosphate 0.75%；Epsom salt 0.03%) in ferment, 30 DEG C culture 5d after, centrifugation thalline obtain supernatant be Crude enzyme liquid.Carry out the detection of NSP enzymes enzyme activity respectively by the crude enzyme liquid to acquisition.
Applicant finally filters out one plant of NSP production of enzyme highest mutant strain, is named as aspergillus niger NSP-5 (Aspergillus niger NSP-5), the xylanase activity 106u/ml that (is shown in Table 1) in the strain fermentation supernatant, than setting out Bacterium improves 96%；Cellulose enzyme activity 4.4u/ml, 91% is improved than going out bacterium germination；1,4 beta-glucanase enzyme activity 41.4u/ml, than Go out bacterium germination and improve 55%；Mannase enzyme activity 1.9u/ml, improve 175% than going out bacterium germination, mutant strain and starting strain Compare, extremely significantly (P ＜ 0.01) improves NSP production of enzyme, achieves unexpected technique effect.
Table 1
Applicant is on July 4th, 2016 by above-mentioned mutant strain aspergillus niger NSP-5 (Aspergillus niger NSP-5 the China typical culture collection center of Wuhan, China Wuhan University) is preserved in, deposit number is CCTCC NO: M2016362。
The 20L ferment tanks of embodiment 3 are verified
Bacterium germination NSP-1 will be gone out and mutant bacteria aspergillus niger NSP-5 is inoculated in identical shake-flask seed culture medium (glucose respectively 10-30g/L, potato 100-200g/L), 30 DEG C, after 200rpm shaking table cultures 20h, zymotic fluid is then transferred to 20L hairs respectively (formula is fermentation tank：Wheat bran 2%；Lactose 0.3%；Corn steep liquor 2%；Ammonium sulfate 0.5%；Potassium dihydrogen phosphate 0.35%；Phosphoric acid Hydrogen dipotassium 0.75%；Epsom salt 0.03%), temperature is controlled at 30 ± 1 DEG C, and pH value is controlled 5.0 ± 0.2, stirring Speed is 600rpm, and after fermentation tank culture 24h, starting to add xylose+glucose reversion syrup, (formula is：Xylose 25%, Glucose 25%, 85% phosphoric acid 5%v/v, 121 DEG C of processing 30min) induction thalline producing enzyme, dissolved oxygen control is in 30-40%, fermentation Time is about 160h, and zymocyte liquid is made.
Above-mentioned zymocyte liquid is centrifuged, takes supernatant, is carried out the detection of NSP enzymes enzyme activity respectively.As a result as shown in Table 2, The fermented supernatant fluid xylanase activity for going out bacterium germination is 99u/ml, cellulose enzyme activity 10.4u/ml, 1,4 beta-glucanase enzyme activity For 37.9u/ml, mannase enzyme activity is 1.5u/ml.Mutant bacteria aspergillus niger NSP-5 fermented supernatant fluid xylanase activity 205u/ml, 107% is improved than going out bacterium germination；Cellulose enzyme activity 17.6u/ml, 69% is improved than going out bacterium germination；Beta glucan Enzyme enzyme activity 67.7u/ml, 78% is improved than going out bacterium germination；Mannase enzyme activity 3.9u/ml, 160% is improved than going out bacterium germination, Each component expression quantity amplification compared with shaking flask is obvious (P ＜ 0.01), achieves unexpected technique effect.
Table 2
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should It is considered as protection scope of the present invention.

Claims (10)

1. aspergillus niger, it is characterised in that its deposit number is CCTCC NO:M2016362.
2. application of the aspergillus niger according to claim 1 in fermenting and producing non-starch polysaccharide enzyme.
3. application according to claim 2, it is characterised in that the non-starch polysaccharide enzyme is zytase, cellulase, Mixture more than one or both of 1,4 beta-glucanase and mannase.
4. a kind of fermentation process for producing non-starch polysaccharide enzyme, it is characterised in that using aspergillus niger as claimed in claim 1 as hair Yeast-like fungi strain.
5. fermentation process according to claim 4, it is characterised in that fermentation process includes shake flask fermentation and fermentation tank is sent out Ferment；
The fermentation medium of the fermentation process includes the mass parts of wheat bran 2；The mass parts of lactose 0.3；The mass parts of corn steep liquor 2； The mass parts of ammonium sulfate 0.5；The mass parts of potassium dihydrogen phosphate 0.35；The mass parts of dipotassium hydrogen phosphate 0.75；The mass of epsom salt 0.03 Part.
6. the fermentation process according to claim 4 or 5, it is characterised in that the fermentation temperature of the shake flask fermentation is 30 DEG C, Fermentation time is 5d.
7. according to the fermentation process described in any one of claim 4 to 6, it is characterised in that the ferment tank includes shaking flask Culture and fermentation tank culture；
The culture medium of the Shaking culture includes glucose 10-30g/L, potato 100-200g/L；
The condition of the Shaking culture is in 30 DEG C, 200rpm shaking table cultures 20h.
8. fermentation process according to claim 7, it is characterised in that the condition of the fermentation tank culture is：30 ± 1 DEG C, pH value is 5.0 ± 0.2, and under conditions of mixing speed is 600rpm, fermentation strain is inoculated in as claimed in claim 5 After cultivating 24h in fermentation medium, xylose+glucose reversion syrup is added, it is 30-40% to control dissolved oxygen, and ferment 160h.
9. fermentation process according to claim 8, it is characterised in that the preparation side of the xylose+glucose reversion syrup Method is：Take xylose 25%, glucose 25%, 85% phosphoric acid 5%v/v, 121 DEG C of processing 30min.
10. the non-starch polysaccharide enzyme that the fermentation process fermentation according to any one of claim 4 to 9 obtains；The non-starch Polysaccharase is zytase, cellulase, mixture more than one or both of 1,4 beta-glucanase and mannase.
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